CZ287157B6 - Defective recombinant adenovirus and pharmaceutical preparation in which said adenovirus is comprised - Google Patents

Abstract

In the present invention there is disclosed a defective recombinant adenovirus containing sequences ITR, a sequence enabling encapsulating and sequence of a heterologous DNA comprising one or several therapeutic genes and/or one or several genes encoding antigen proteins whereby in said adenovirus the E1 and at least one of the E2, E4 and L1 - L5 are non-functional. In the present invention there is also claimed a pharmaceutical preparation in which said adenovirus is comprised as active substance.

Description

A defective recombinant adenovirus and a pharmaceutical composition comprising the adenovirus

Technical field

The invention relates to novel viral vectors and to a process for their preparation and use in gene therapy. The invention also relates to pharmaceutical compositions comprising these viral vectors. In particular, the invention relates to recombinant adenoviruses used as gene therapy vectors.

BACKGROUND OF THE INVENTION

Gene therapy consists in correcting some deficiencies and abnormalities (mutations, aberrant expression, etc.) by introducing genetic information into the affected cell or organ. This genetic information can be introduced either in vitro into a cell extracted from an organ, whereby the modified cell is then reintroduced into the organism, or in vivo directly into the respective tissue. With respect to this second method, there are various techniques from which individual transfection techniques may be mentioned, including the use of DNA and DEAE-dextran complexes (Pasgano et al., J. Virol. 1 (1967) 891), DNA and envelope proteins (Kaneda and et al., Science 243 (1989) 375), DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), the use of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), etc. Recently, the use of viruses as gene transfer vectors has been shown to be a promising alternative to these physical transfection techniques. In this context, various viruses have been tested to determine their ability to infect different cell populations. In particular, retroviruses (RSV, HMS, MMS, etc.), HSV virus, adeno-associated viruses and adenoviruses were tested in this way.

Of these viruses, in particular, adenoviruses have been shown to have some interesting properties in terms of their usefulness in gene therapy. In particular, adenoviruses have a fairly broad host spectrum, are capable of infecting immobile cells, do not associate with the genome of infected cells, and have not been associated with significant pathologies in humans to date.

Adenoviruses are linear double stranded DNA viruses of about 36 kb in length. In particular, their genome comprises an inverted repeat sequence (ITR) at their end, an encapsulation sequence, early genes and late genes (see Figure 1). The basic late genes are genes L1 to L5.

In view of these properties, adenoviruses have already been used for gene transfer in vivo. To this end, various adenovirus-derived vectors have been prepared in which different genes have been incorporated (beta-gal, OTC, alpha-ΙΑΤ, cytokines, etc.). In each system so constructed, the adenovirus was modified to be incapable of replicating in the infected cell. Thus, adenoviruses with deletions of the E1 (Ela and / or Elb) and E3 regions, where appropriate, heterologous DNA sequences are inserted at these regions (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury) et al., Gene 50 (1986) 161). However, the vectors described so far have numerous drawbacks that limit their use in gene therapy. In particular, all these vectors contain numerous viral genes whose expression in vivo is not desirable in gene therapy. In addition, these vectors do not allow the incorporation of DNA fragments of considerable length, which may be necessary in some applications.

SUMMARY OF THE INVENTION

The invention makes it possible to eliminate these drawbacks. Recombinant gene therapy adenoviruses that are capable of efficiently transmitting in vivo DNA (up to 30 kb), expressing this DNA in vivo in a high and stable manner are eliminated, eliminating any risk of viral protein production, viral transfer, pathogenicity, etc. In particular, it was found that

That it is possible to greatly reduce the genome size of the adenovirus without inhibiting the formation of encapsulated virus particles. This is surprising given that for some other viruses, such as retroviruses, it has been observed that some sequences distributed along the genome were necessary for effective encapsulation of viral particles. As a result, the realization of vectors having significant internal deletions was severely restricted. The invention also shows that the suppression of a substantial portion of the viral genes does not prevent the formation of such a viral particle. In addition, the recombinant adenoviruses thus obtained retain, in spite of significant modifications in their genomic structure, their advantageous properties, in particular their strong infectious ability and stability in vivo.

The vectors of the invention are particularly advantageous since they allow the incorporation of the desired DNA sequences of considerable length. Thus, it is possible to insert a gene having a length of more than 30 kb. This is of particular interest in some pathologies whose treatment requires the coexpression of several genes or the expression of very long genes. For example, in the case of muscular dystrophy, it has not been possible to transfer DNA corresponding to the native gene responsible for this pathological condition (the dystrophin gene) due to its considerable length (14 kb).

The vectors of the invention are also very advantageous in that they have very few functional viral regions and therefore the risk associated with using the virus as a vector in gene therapy (immunogenicity, pathogenicity, transmission, replication, recombination, etc.) Is greatly reduced or completely suppressed. .

Thus, the invention provides viral vectors that are particularly adapted to transmit and express in vivo the desired DNA sequences.

Thus, a first aspect of the invention relates to a defective recombinant adenovirus comprising:

- ITR sequence,

the encapsulation sequence, and

a heterologous DNA sequence comprising one or more therapeutic genes and / or one or more genes encoding antigenic proteins, wherein in this adenovirus the E1 gene and at least one of the E2, E4 and L1-L5 genes are non-functional.

The term "defective adenovirus" within the scope of the invention refers to an adenovirus that is not able to autonomously replicate in the target cell. Thus, in general, the genome of the defective adenovirus of the invention is devoid of at least those sequences which are necessary for the replication of the virus in the infected cell. These regions can either be removed (wholly or partially) or rendered non-functional or replaced by other sequences, particularly heterologous DNA sequences.

Inverted repeat sequences (ITRs) are the origin of adenovirus replication. These sequences are located at the ends 3 'and 5' of the viral genome (see Figure 1), from where they can be easily isolated by classical molecular biology techniques. The nucleotide sequence of the inverted human adenovirus repeat sequences (particularly Ad2 and Ad5 serotypes) as well as canine adenoviruses (especially CAV1 and CAV2) are described in the literature. For example, for an Ad5 adenovirus, the left ITR sequence corresponds to a region comprising nucleotides 1 to 103 of the genome.

The encapsulation sequence (also referred to as the Psi sequence) is necessary for encapsulation of viral DNA. Thus, this region must be present in order to prepare the defective recombinant virus of the invention. This encapsulation sequence is located in the adenovirus genome between the left inverted repeat sequence (5 ') and the E1 gene (see Figure 1). This sequence can be isolated or synthesized artificially by classical molecular biology techniques. The nucleotide sequence of the encapsulation sequence of human adenoviruses (particularly Ad2 and Ad5 serotypes) as well as canine adenoviruses (especially CAV1 and CAV2) is described in the relevant literature. As far as it goes

For example, an Ad5 adenovirus corresponds to the encapsulation sequence of a region comprising nucleotides 194 to 358 of the genome.

There are various adenovirus serotypes whose structure and properties vary somewhat. However, these viruses have a comparable genetic organization and the instructions described in this patent application can be readily utilized by one skilled in the art for any type of adenovirus.

The adenoviruses of the invention may be of human, animal or mixed (human and animal) origin.

With respect to adenoviruses of human origin, it is preferable to use adenoviruses classified in Group C. Even more preferably, of the various serotypes of human adenovirus, adenoviruses of type 2 or 5 (Ad 2 or Ad 5) are used in the invention.

As mentioned above, the adenoviruses of the invention may also be of animal origin, or may comprise sequences of zadenoviruses of animal origin. Indeed, the Applicant has shown that adenoviruses of animal origin are capable of infecting human cells very efficiently and that they are unable to multiply in the human cells in which they have been tested (see patent application FR 93 05954). The Applicant has also shown that adenoviruses of animal origin are not transcomplemented with adenoviruses of human origin, eliminating the risk of recombination and propagation in vivo in the presence of human adenovirus, which could lead to the formation of an infectious particle. Thus, the use of adenoviruses or regions of adenoviruses of animal origin is particularly advantageous since the risks associated with the use of the virus as a vector in gene therapy are further reduced.

The adenoviruses of animal origin useful in the present invention may be of canine, bovine, murine (for example: Mávl, Beard et al., Virology 75 (1990) 81), sheep, porcine, avian or also monkey (for example: SAV) origin. Among the avian adenoviruses, particular mention may be made of serotypes 1 to 10 available in the ATCC, for example the strains Phelps (ATCC VR-432), fontes (ATCC VR-280), P7-A (ATCC VR-827), IBH-2A (ATCC VR-828 ), J2-A (ATCC VR-829), T8-A (ATCC VR-830), K-11 (ATCC VR-921) or also strains referred to as ATCC VR-831. Among the bovine adenoviruses, various known serotypes can be used, and in particular the serotypes available in ATCC (types 1 to 8) under the designation ATCC VR-313, 314, 639-642, 768 and 769. Mouse FL adenoviruses (ATCC VR-550) and E20308 (ATCC VR-528), sheep adenovirus type 5 (ATCC VR-1343) or type 6 (ATCC VR-1340), porcine adenovirus 5359, or simian adenoviruses, such as, but not limited to, the adenoviruses listed in the ATCC under VR-591-594, 941-943, 195-203, etc ..

Advantageously, of the various adenoviruses of animal origin, adenoviruses or regions of adenoviruses of canine origin, and in particular all CAV2 adenoviruses (e.g. manhattan strain or A26 / 61 (ATCC VR-800)), are used in the invention. Dog adenoviruses have been the subject of numerous structural studies. Thus, complete restriction maps of CAV1 and CAV2 adenoviruses (Spibey et al. J. Gen. Virol. 70 (1989) 165) have been described and the Ela and E3 genes as well as the ITR sequences have been cloned and sequenced (see in particular Spibey et al., Virus). Res. 14 (1989) 241, Linna virus Res. 23 (1992) 119, WO 91/11525).

As mentioned above, the adenoviruses of the invention contain a heterologous DNA sequence. This heterologous DNA sequence refers to any DNA sequence introduced into a recombinant virus whose transfer to a target cell and / or expression in a target cell is desired.

The heterologous DNA sequence may comprise one or more therapeutic genes encoding antigenic peptides.

Therapeutic genes that can be so transferred are all genes whose transcription or translation in the target cell generates products having a therapeutic effect.

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In particular, they may be genes encoding protein products having a therapeutic effect. The protein product thus encoded may be a protein, peptide, amino acid, etc. This protein product may be homologous to the target cell (i.e., a product that is normally expressed in the target cell if the cell exhibits no pathology). In this case, for example, the expression of the protein makes it possible to improve the under-expression in the target cell of the protein or to replace the expression of the inactive or low-active protein by the expression of the active protein. The therapeutic gene may also encode a mutant of a cellular protein having increased stability, modified activity, etc. The protein product may also be heterologous to the target cell. In this case, the expressed protein may, for example, complement and ameliorate cell malfunction, thereby allowing the cell to be able to combat a pathological condition.

Among the therapeutic products mentioned herein are, in particular, enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF, etc. (FR 9203120), growth factors, neurotransmitters or their precursors or synthesis enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, etc., apolipoproteins: ApoAI, ApoAIV, ApoE, etc. (FR 93 05125), dystrophm or minidystrophin (FR 9111947), tumor suppressor genes: p53, Rb, Rapi A, DCC, k-rev, etc. (FR 93 04745), genes encoding factors involved in coagulation: factors VII, VIII, IX, etc.

The therapeutic gene may also be an antisense gene or an antisense sequence, the expression of which in the target cell makes it possible to regulate gene expression or transcription of cellular mRNAs. Such sequences may, for example, be transcribed in the target cell into complementary RNA of cellular mRNAs and thus block their translation into proteins by the technique described in EP 140 308.

As mentioned above, the heterologous DNA sequence may also contain one or more genes encoding an antigenic peptide capable of generating an immune response in humans. In this specific embodiment, the invention thus makes it possible to realize vaccines enabling to make a human immune, especially against microorganisms or viruses. These may in particular be specific antigen peptides of epstein barr virus, HIV virus, hepatitis B virus (EP 185 573), rabies virus or also specific antigen peptides of tumors (EP 259 212).

Generally, the heterologous DNA sequence also comprises sequences allowing expression of the therapeutic gene and / or the gene encoding the antigenic peptide in the infected cell. These may be sequences which are naturally responsible for the expression of the gene of interest in case the sequences are capable of functioning in the infected cell. They may also be sequences of different origins (responsible for the expression of other proteins or even synthetic sequences). In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences from the genomes of the cell to be infected. Similarly, they may be promoter sequences from the genome of the virus, including the adenovirus used. In this context, for example, promoters of the E1A, MLP, CMV, RSV, etc. genes may be modified. In addition, these expression sequences may be modified by adding activation sequences, regulatory sequences, etc. If the gene insert does not contain an expression sequence, it may be inserted into the defective genome virus after such a sequence.

Moreover, the heterologous DNA sequence may also contain, in particular upstream of the therapeutic gene, a signal sequence directing the synthesized therapeutic product into the secretory pathways of the target cell. The signal sequence may be a natural signal sequence of the therapeutic product, although it may also be any other functional signal sequence or an artificial signal sequence.

As mentioned above, the vectors of the invention have at least one of the E2, E4, L1-L5 genes non-functional. The viral gene contemplated may be rendered inoperative by any known technique

Used for this purpose, in particular by suppressing, substituting, deleting or adding one or more bases to the gene or genes under consideration. These modifications can be achieved in vitro (on isolated DNA) or in situ, for example using genetic engineering techniques or also by treatment with mutagenic agents.

These mutagenic agents include, for example, physical agents such as energy rays (X-rays, gamma rays, ultraviolet rays, etc.), or chemical agents capable of reacting with various functional bases of DNA bases, such as alkylating agents / ethyl methanesulfonate (EMS). , N-methyl-N'-nitro-N-nitrosoguanidine, N-nitroquinoline 1-oxide (NQO) /, bialkylating agents, intercalating agents, etc.

By deletion is meant within the scope of the invention any suppression of the gene of interest. In particular, it may be all or part of the coding region of said gene and / or all or part of the promoter region of transcription of said gene. Said suppression may be accomplished by digestion with the appropriate restriction enzymes, then ligation using classical molecular biology techniques as illustrated in the examples below.

Genetic modification can also be achieved by gene digestion, for example according to the protocol originally described by Rothstein / Meth. Enzymol. 101 (1983) 202]. In this case, part or all of the coding sequence is preferably disrupted to allow replacement by homologous recombination of the genomic sequence with a non-functional or mutant sequence.

Said genetic modification or genetic modifications may be located within or outside such coding region of the gene of interest, for example, regions responsible for expression and / or transcriptional regulation of said genes. Thus, the non-functional character of said genes may be manifested by the production of inactive protein caused by structural or conformational modifications, by the production of a protein with impaired activity, or by the production of natural protein to a reduced extent or according to the desired regulatory regimen.

Some changes, such as point mutations, are of such a nature that they can be corrected or attenuated by cellular mechanisms. Such genetic changes are of limited importance on an industrial scale. It is therefore particularly advantageous if said non-functional character is perfectly segregatingly stable and / or irreversible.

Preferably, the gene is non-functional due to a partial or complete deletion.

Preferably, the defective recombinant adenoviruses of the invention are devoid of late adenovirus genes.

A particularly preferred embodiment of the invention comprises a defective recombinant adenovirus comprising:

- ITR sequence,

- encapsulation sequence,

heterologous DNA sequence, and

- the region carrying the E2 gene or part thereof.

Another particularly preferred embodiment of the invention comprises a defective recombinant adenovirus comprising:

- ITR sequence,

- encapsulation sequence,

heterologous DNA sequence, and

- the region carrying the E4 gene or part thereof.

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Also in a particularly preferred embodiment of the invention, the vectors of the invention additionally have an E3 gene whose function is under the control of a heterologous promoter. More preferably, said vectors have a portion of the E3 gene allowing expression of the gp19K protein.

A defective method for preparing said adenoviruses is to transfect DNA of a defective recombinant virus prepared in vitro (either by ligation or in the form of a plasmid) into a competent cell line, i.e. carrying in the trans position all functions necessary to complement the defective virus. These functions are preferably integrated in the genome of the cell, thereby avoiding the risk of recombination and conferring increased stability to the cell line. The preparation of such cell lines is described in the examples below.

A second method for preparing said defective recombinant adenoviruses is to simultaneously transfect DNA of the defective recombinant virus prepared in vitro (either by ligation or in the form of a plasmid) and helper virus DNA into the respective cell line. In this method, it is not necessary to have a competent cell line capable of complementing all defective functions of recombinant adenoviruses. Part of these functions are in fact complemented by said helper virus. Thus, this helper virus must itself be defective and the cell line carries at the trans position necessary for its complementation. This preparation of defective recombinant adenoviruses is also illustrated in the following examples.

Among the cell lines useful in this second method of preparing adenoviruses, in particular, the human germline 293 cell line, KB cell, Hela cell, MDCK, GHK, etc. are mentioned (see the Examples below).

Vectors that have been multiplied are then isolated, purified and amplified using conventional molecular biology techniques.

The invention also relates to a pharmaceutical composition comprising one or more defective recombinant adenoviruses as described above. The pharmaceutical compositions of the invention may be formulated in a form suitable for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal or other suitable administration.

Preferably, the pharmaceutical composition comprises carriers that are pharmaceutically acceptable for an injectable composition. In particular, they may be solutions of salts (sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of such salts) in sterile, isotonic form, or compositions in dried, in particular lyophilized form, of which, after addition of sterilized water or physiological serum, provides injectable solutions.

The doses of virus used for injection may depend on various parameters, in particular on the route of administration used, the pathological condition being treated, the gene to be expressed, or also the duration of treatment envisaged. Generally, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 10 ⁴ and 10 ¹⁴ pfu / ml, preferably 10 ⁶ to 10 ^I0 pfu / ml. The term pfu (plaque forming unit) corresponds to the infectious ability of the virus solution and is determined by infection of the cell culture in question, generally measured after 5 days by determining the number of infected cell areas. The techniques that can be used to determine the pfu titre of a virus solution are well documented in the relevant literature.

Depending on the inserted heterologous DNA sequence, the adenoviruses of the invention can be used to treat or prevent a number of pathological conditions including genetic diseases (dystrophy, cystic fibrosis, etc.), neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, ALS, etc.), pathological cancers conditions associated with coagulation disorders

-6US 287157 B6 or dyslipoproteinemia, pathological conditions associated with viral infections (hepatitis, AIDS, etc.), etc.

Overview of the drawings

The invention will be further elucidated with reference to the accompanying drawings, in which Figure 1 shows the genetic organization of the Ad5 adenovirus. Based on the data provided herein, the complete Ad5 adenovirus sequence is available, and this sequence allows one of skill in the art to select or create any restriction site and thereby isolate any region of the genome.

Giant. 2 shows a restriction map of the CAV2 adenovirus (Manhattan strain, according to Spilbey et al., Supra).

Giant. 3 shows the construction of a defective virus according to the invention by ligation.

Giant. 4 shows the construction of a recombinant virus carrying the E4 gene.

Giant. 5 shows the construction of a recombinant virus carrying the E2 gene.

Giant. 6 shows the construction and display of plasmid pPY32.

Giant. 7 shows a representation of plasmid pPY55.

Giant. 8 shows a representation of plasmid p2.

Giant. 9 is an illustration of an intermediate plasmid used to construct plasmid pITRL5-E4.

Giant. 10 shows a representation of plasmid pITRL-E4.

DETAILED DESCRIPTION OF THE INVENTION

In these examples, the invention will be illustrated in more detail by way of specific examples, which are intended to be illustrative only, and are not intended to limit the scope of the invention as defined by the claims.

General techniques of molecular biology

Classical methods used in molecular biology, such as preparative plasmid DNA extraction, plasmid DNA centrifugation in cesium chloride gradient, agar or acrylamide gel electrophoresis, purification of DNA fragments by electroelution, proteins with phenol or phenol / chloroform, precipitation of DNA in ethanol or isopropanol transformations in Escherichia coli, etc. are well known to those skilled in the art and are sufficiently described in the literature / Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982, Ausubel FM et al. (publ.), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987 /.

Plasmids of the pBR322 type, pUC, and M13 series phages are commercially available (Bethesda Research Laboratories).

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For ligation, DNA fragments can be separated according to their size by agar or acrylamide gel electrophoresis, extracted with phenol or phenol / chloroform, precipitated with ethanol, and then incubated in the presence of phage T4 DNA ligase (Biolabs) as recommended by the supplier.

The addition of the proeminent ends 5 'can be performed using the Klenow fragment of Escherichia coli DNApolymerase I (Biolabs) according to the supplier's instructions. The destruction of the proeminent ends 3 'is performed in the presence of phage T4 DNA polymerase (Biolabs) used according to the manufacturer's recommendations. The destruction of the proeminent ends 5 'is effected by gentle treatment with S1 nuclease.

Controlled in vitro nutagenesis using synthetic oligodeoxynucleotides can be accomplished by the method developed by Taylor et al. Nucleic Acids Res. 13 (1985) 8749-8764] using a kit distributed by Amersham.

Enzymatic amplification of DNA fragments by a technique called PCR / Polymerase-Catalyzed Chain Reaction, Saiki R. K. et al., Science 230 (1985) 1350-1354, Mullis K. B. and Faloon F. A., Meth. Enzyme. 155 (1987) 335-350] may be performed using a DNA thermal cycler system (Perkin Elmer Cetus) according to the manufacturer's instructions.

Verification of nucleotide sequences can be performed by the method developed by Sanger et al. /Why. Nati. Acad. Sci. USA, 74 (1997) 5463-5467] using a kit distributed by Amersham.

Used cell lines

The following cell lines may be used in the examples below:

Cell line 293 from human embryonic kidney (Graham et al., J. Gen. Virol. 36 (1977) 59). In particular, this cell line contains, in its genome form, the left-hand part of the human Ad5 adenovirus genome (12%).

- Human KB cell line: from human epidermal carcinoma. This cell line, as well as the conditions allowing its culture, are available at ATCC (reference designation CCL17).

- Hela human cell line: originated from human epithelial carcinoma. This cell line is available from ATCC (CCL2 reference designation). Similarly, there are conditions available for culturing this cell line.

- Canine MDCK cell line: MDCK cell culture conditions have been described in particular by Macatney et al. in Science 44 (1988) 9.

Gm DBP6 cell line (brough et al., Virology 190 (1992) 624). This cell line consists of Hela cells carrying the adenovirus E2 gene under the control of MMTV LTR (long terminal repeat).

Example 1

This example demonstrates the viability of a recombinant adenovirus devoid of a substantial portion of the viral genes. To this end, a series of deletion mutants were constructed in an adenovirus by in vitro ligation, each of these mutants being co-transfected with helper virus into KB cells. Since these cells do not allow the virus to be defective in the case of E1, transcomplementation is directed to the E1 region.

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Various deletion mutants were prepared from Ad5 adenovirus by digestion and then in vitro ligation. To this end, the Ad5 viral DNA of the Ad5 virus is isolated by the technique described by Lipp et al. (J. Virol. 63 (1989) 5133), then digested with various restriction enzymes (see FIG. 3), and the product obtained by this digestion is ligated in the presence of T4DNA ligase. The size of the individual deletion mutants is then checked on an SDS-agar gel (0.8%). These deletion mutants are then mapped (see Figure 3). These individual mutants contain the following regions:

mtl: ligation between Ad5 adenovirus fragments 0-20642 (SauI) and (Sau) 33797-35935, mt2: ligation between Ad5 adenovirus fragments 0-19549 (NdeI) and (NdeI) 31089-35935, mt3: ligation between Ad5 adenovirus fragments 0 -10754 (AaII) and (AatII) 25915-35935, mt4: ligation between Ad5 adenovirus fragments 0-1131 l (MluI) and (MluI) 24392-35935, mt5: ligation between Ad5 adenovirus fragments 0-9462 (SalI) and ( XhoI 29791-35935, mt6: ligation between Ad5 0-5788 (XhoI) and (XhoI) 29791-35935 adenovirus fragments 0-3665 (SphI) and (SphI) 31224-35935.

Each of the mutants thus prepared was transfected together with Ad.RSVbetaGal viral DNA (Stratford-Perricaudet et al., J. Clin. Invest. 90 (1992 626) into KB cells in the presence of calcium phosphate, 8 days after transfection, cells were isolated and supernatants cultures were amplified over KB cells until a stock was obtained for each transfection, and episomal DNA was isolated and separated on a cesium chloride gradient from each sample, in each case two distinct bands of virus were observed and isolated and analyzed . heaviest corresponds viral DNA Ad.RSVbetaGal lightest corresponds DNA of the recombinant virus generated by ligation (Fig. 3). the titer of the latter case is about 10 ⁸ pfu / ml.

Using the same methodology, a second series of deletion mutants were constructed in adenovirus by in vitro ligation. These various mutants comprise the following regions:

mt8: ligation between fragments 0-10178 (ApaI) Ad RSVbetaGal and (ApaI) 31909-35935 Ad5. mt9: ligation between fragments 0-10178 (BglII) of Ad RSVbetaGal and (BamHI) 21562-35935 Ad5.

These mutants carrying the LacZ gene under the control of the RSV LTR promoter are then cotransfected into 293 cells in the presence of H2d1880 viral DNA (Weinberg et al., J. Virol. 57 (1986) 833), which is subjected to deletion in the E4 region. According to this second technique, transcomplementation is directed to E4 and no longer to E1. As mentioned above, this technique thus allows the generation of recombinant viruses having only the E4 region as a viral gene.

Example 2

This example describes the preparation of a defective recombinant adenovirus according to the invention by co-transfecting helper virus and recombinant virus DNA incorporated into a plasmid.

To this end, a plasmid carrying the Ad5 adenovirus junction ITR, the encapsulation sequence, the E4 gene under the control of its own promoter, and the LacZ gene under the control of the RSV LTR promoter were constructed as a heterologous gene (FIG. 4). This plasmid, referred to as pE4Gal, was obtained by cloning and ligation of the following fragments (see Figure 4):

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HindIII-SacII fragment from plasmid pFG144 (Graham et al., EMBO J. 8 (1989) 2077). This fragment carries the ITR sequences of the Ad5 adenovirus at the beginning and at the end, and the encapsulation sequence: HindIII (34920) -SacII fragment (352).

- Ad5 adenovirus fragment contained between SacII sites (located at base pair 3827) and PstI (located at base pair 4245).

A fragment of pSP 72 (Promega) contained between the PstI (pb 32) and SalI (pb 34) sites.

The XhoI-XbaI fragment of plasmid pAdLTR GalIX described by Stratford-Perricaudet et al. (JCI 90 (1992) 626). This fragment carries the LacZ gene under the control of the RSV LTR.

- XbaI fragment (pb 40) -NdeI (pb 2379) of plasmid pSP 72.

- NdeI fragment (pb 31089) - HindIII (pb 34930) of Ad5 adenovirus. This fragment, located at the right end of the genome of the Ad5 adenovirus, contains the E4 region under the control of its own promoter. It was cloned at the NdeI (2379) sites of plasmid pSP 72 and HindIII of the first fragment.

This plasmid was obtained by cloning various fragments in the designated region of plasmid pSP 72. Of course, equivalent fragments can be obtained from other sources by one skilled in the art.

This plasmid pE4Gal is then transfected together with the H2d1880 virus DNA into 293 cells in the presence of calcium phosphate. The recombinant virus is then prepared as described in Example 1. This virus carries the only viral gene, the E4 gene of the Ad5 adenovirus (FIG. 4). Its genome is about 12 kb in size, allowing heterologous DNA of considerable size (up to 20 kb) to be inserted. Thus, one skilled in the art can easily replace the LacZ gene with any other therapeutic gene from the plurality of genes already mentioned above. In addition, this virus carries some sequences derived from plasmid pSP 72, which may optionally be eliminated by conventional molecular biology techniques.

Example 3

This example describes the preparation of another defective recombinant adenovirus of the invention by co-transfection of helper virus and recombinant virus DNA incorporated into the plasmid.

To this end, a plasmid carrying an Ad5 adenovirus junction ITR, an encapsulation sequence, an Ad2 adenovirus E2 gene under the control of its own promoter and a heterologous LacZ gene under the control of the RSV LTR promoter is constructed (FIG. 5). This plasmid referred to as pE2Gal was obtained by cloning and ligation of the following fragments (see Figure 5):

HindIII-SacII fragment derived from plasmid pFG144 (Graham et al., EMBO J. 8 (1989) 2077). This fragment carries at the beginning and the end of the ITR of the Ad5 adenovirus and the encapsulation sequence: HindIII (34920) -SacII fragment (352). It was cloned with the following fragment at the HindIII (16) -PstI (32) site level of plasmid pSP 72.

- Ad5 adenovirus fragment contained between SacII sites (located at base pair 3827) and PstI (located at base pair 4245). This fragment was cloned at the SacII site of the preceding fragment and the PstI (32) site of plasmid pSP 72.

A fragment of plasmid pSP 72 (Promega) contained between the PstI (pb 32) and SalI (pb 34) sites.

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The XhoI-XbaI fragment of plasmid pAdLTR GalIX described by Stratford-Perricaudet et al. (JCI 90 (1992) 626). This fragment carries the LacZ gene under the control of the RSV LTR. It was cloned at the SalI (34) and XbaI sites of plasmid pSP 72.

- Plasmid fragment of pSP 72 (Promega) contained between the XbaI sites (pb 34) and BamHI (pb 46).

- BamHI fragment (pb 21606) - SmaI (pb 27339) of Ad2 adenovirus. This Ad2 genome fragment contains the E2 region under the control of its own promoter. It was cloned at the BamHI (46) and EcoRV sites of plasmid pSP 72.

- EcoRV fragment (pb 81) - HindIII (pb 16) of plasmid pSP 72.

This plasmid was obtained by cloning various fragments in the designated region of plasmid pSP 72. Of course, equivalent fragments can be prepared by one skilled in the art from other sources.

Plasmid pE2Gal is then transfected together with DNA of the H2d1802 virus depleted of the E2 regions (Rice et al., J. Virol. 56 (1985) 767) into 293 cells in the presence of calcium phosphate. The recombinant virus is then prepared as described in Example 1. This virus carries the Ad2 adenovirus E2 gene as the only viral gene (FIG. 5). Its genome is about 12 kb in size, which allows the insertion of heterologous DNA of considerable size (up to 20 kb). Thus, one skilled in the art can easily replace the LacZ gene with any other therapeutic gene from the plurality of genes already mentioned above. Moreover, this virus contains some sequences derived from the intermediate plasmid, which can be removed, if necessary, by conventional molecular biology techniques.

Example 4

This example describes the construction of cell lines complementary to the E1, E2 and / or E4 regions of adenoviruses. These cell lines allow the construction of recombinant adenoviruses of the invention subjected to deletion in these regions without the participation of helper virus. These viruses are obtained by recombination in vivo and may contain significant heterologous sequences.

In the described, potentially cytotoxic regions E2 and E4 are placed under the control of the inducible promoter: LTR MMTV (Pharmacia) which is induced by dexamethasone, either in native form or in the minimal form described in PNAS 90 (1993) 5603; or the tetracycline suppressible system described by Gossen and Bujard (PNAS 89 (1992) 5547). It goes without saying that other promoters may be used, in particular LTTV MMTV variants carrying, for example, regulatory heterologous regions (particularly the enhancer region). The cell lines of the invention were prepared by transfection in the presence of calcium phosphate of the corresponding cells with a DNA fragment carrying the indicated genes (adenovirus and / or glucocordicoid receptor gene) under the control of the transcriptional promoter and terminator (polyadenylation site). The terminator may be either a natural terminator of the transfected gene or a different terminator, such as the SV40 early mediator terminator. Preferably, the DNA fragment also carries a gene allowing selection of transformed cells and, for example, a genicitin resistance gene. This resistance gene may also be carried by a different DNA fragment co-transfected with the first fragment.

After transfection, the transformed cells are subjected to selection and their DNA is analyzed to verify the integration of the DNA fragment into the gemon.

-11EN 287157 B6

This technique makes it possible to obtain the following cell lines:

1. 293 cells containing a gene from the 72K region of the E2 adenovirus Ad5 under the control of MMTV LTRs;

2. 293 cells containing a gene from the 72K region of the E2 region of the Ad5 adenovirus under the control of the MMTV MMR and a glucocorticoid receptor gene;

3. 293 cells containing a gene from the 72K region of the E2 region of the Ad5 adenovirus under the control of MMTV LTR and the E4 region under the control of MMTV LTR,

4. 293 cells containing the gene from the 72K region of the Ad2 adenovirus E2 under the control of the MMTV MMR, the E4 region under the control of the MMTV MMTV, and the glucocorticoid receptor gene;

5. 293 cells having the E4 region under control of the MMTV LTR;

6. 293 cells having the E4 region under control of the MMTV LTR and a glucocorticoid receptor gene;

7. DBP6 gm cells having E1A and E1B regions under the control of their own promoter; and

8. DBP6 gm cells having the E1A and E1B regions under the control of their own promoter and the E4 region under the control of the MMTV LTR.

Example 5

This example describes the preparation of defective recombinant adenoviruses of the invention whose genome has been deleted for the E1, E3 and E4 genes. According to a preferred embodiment illustrated in this example and in Example 3, in particular, the genome of the recombinant adenoviruses of the invention is modified in such a way that at least the E1 and E4 genes are non-functional. In particular, such adenoviruses have the ability to incorporate significant heterologous genes. Otherwise, these vectors have increased safety due to the deletion of the E4 region that is involved in regulating expression of late genes, stability of late coat RNAs, extinction of host cell protein expression, and viral DNA replication efficiency. Thus, these vectors have very limited transcript noise and very limited expression of viral genes. Finally, it is a particularly advantageous fact that these vectors can be produced at concentrations comparable to those obtainable in wild-type adenoviruses.

These adenoviruses were prepared from the plasmid pPY55 carrying the right modified part of the Ad5 adenovirus genome, either by co-transfection with a helper plasmid (see also Examples 1, 2 and 3), or using a complementing cell line (Example 4).

5.1 Construction of plasmid pPY55

a) Construction of plasmid pPY32

AvrII-BcII fragment of plasmid pFG144 / F. L. Graham et al. EMBO J. 8 (1989) 2077-2085] corresponding to the right end of the Ad5 genome of the adenovirus was first cloned between the XbaI and BamHI sites of the pIC19H vector prepared from the dam- structure. This generates the plasmid pPY23. An important characteristic of plasmid pPY23 is that the SalI site derived from the cloning set of sites of the pIC19H vector remains single and is located next to the right end of the Ad5 adenovirus genome. The HaelII-SalI fragment of plasmid pPY23, which contains the right end of the Ad5 adenovirus genome from the HaelII site located at position 35614, is then cloned between the EcoRV and XhoI sites of the pIC20H vector to generate plasmid pPY29. An interesting characteristic of this plasmid is that the XbaI and ClaI sites derived from the cloning set of the pIC20H vector sites are

-12GB 287157 B6 located next to the EcoRV / HaelII junction resulting from cloning. In addition, this junction modifies the nucleotide structure immediately adjacent to the ClaI site, which has now become methylated in the dam + structure. The XbaI (30470) -MaeII (32811) fragment of the Ad5 genome is then cloned between the XbaI and ClaI sites of plasmid pPY29 prepared from the dam- structure, generating plasmid pPY30. The SstI fragment of plasmid pPY30, which corresponds to the Ad5 genome sequence from the SstI site at position 30556 to the right end, is then cloned between the SstI sites of the vector pIC20H, generating plasmid pPY31 whose restriction map with the insert located between HindIII sites is shown in FIG. .

Plasmid pPY32 was obtained after partial digestion of plasmid pPY31 with BblII, complete digestion with BamHI and subsequent ligation. Thus, the plasmid pPY32 corresponds to the Ad5 genome deletion located between the BamHI site of plasmid pPY31 and the BblII site located at position 30818. The restriction map of the HindIII fragment of plasmid pPY32 is shown in Figure 6. The characteristic of plasmid pPY32 is that it has single SalI and XbaI sites.

b) Construction of plasmid pPY47

The BamHI (21562) -XbaI (28592) fragment of the Ad5 adenovirus genome was first cloned between the BamHI and XbaI sites of pC19H vector prepared from the dam- structure, generating plasmid pPY17. Thus, this plasmid contains a HindIII (26328) -BglII (28133) fragment of the Ad5 adenovirus genome that can be cloned between the HindIII and BglII sites of the pIC20R vector to generate plasmid pPY34. One characteristic of this plasmid is that a BamHI site derived from a cloning set of sites is located in close proximity to the HindIII (26328) site of the Ad5 genome.

The BamHI (21562) -HindIII (26328) fragment of the Ad5 adenovirus genome derived from plasmid pPY17 was then cloned between the BamHI and HindIII sites of plasmid pPY34, generating plasmid pPY39. A BamHI-XbaI fragment of plasmid pPY39 prepared from the dam- structure containing a portion of the Ad5 adenovirus genome contained between the BamHI (21562) and BglII sites (28133) was then cloned between the BamHI and Xball sites of the pIC19H vector prepared from the dam- structure. whose interesting characteristic is that the SalI site originating from the cloning set of sites is located near the HindIII site (Fig. 7).

c) Construction of plasmid pPY55

The SalI-XbaI fragment of plasmid pPY47 prepared from the dam- and containing a portion of the Ad5 adenovirus genome starting from the BamHI site (21562) and ending with the BglII site (28133) was cloned between the SalI and XbaI sites of plasmid pPY32, generating plasmid pPY55. This plasmid is directly used to obtain a recombinant adenovirus in which there has been a deletion in at least the E3 region (deletion between the BglII sites located at positions 28133 and 30818 of the Ad5 genome) and the E4 region in its integrity (deletion between the Maell sites (32811) and HaelII (35614) of the Ad5 adenovirus genome (FIG. 7).

5.2. Preparation of an adenovirus comprising at least one deletion in the E4 region and preferably at least in the E1 and E4 regions

a) Preparation of transfections into 293 cells together with helper virus E4

The essence of this preparation consists in a transcomplementation between a "helivirus" expressing the E4 region and a recombinant virus in which at least the E3 and E4 regions have been deleted. These viruses are obtained by in vitro ligation or after in vivo recombination according to the following strategies:

-13GB 287157 B6

i) Both the Ad-dl324 virus DNA (Thimmappaya et al. Cell 31 (1982) 543) and the plasmid pPY55 digested with BamHI were first ligated in vitro and then transfected together with the plasmid pEAGal (described in the example). 2) into 293 cells.

ii) Ad-dl324 virus DNA digested with EcoRI and plasmid pPY55 digested with BamHI were co-transfected with plasmid pE4Gal into 293 cells.

iii) Both the Ad5 adenovirus DNA and the plasmid pPY55 digested with BamHI were ligated and then transfected together with the plasmid pE4Gal into 293 cells.

iv) Ad5 Adenovirus DNA digested with EcoRI and plasmid pPY55 digested with BamHI were transfected together with pEAGa1 into 293 cells.

Strategies (i) and (ii) allow the generation of recombinant adenovirus with deletions in the E1, E3 and E4 regions; strategies iii) and iv) allow the generation of recombinant adenovirus with deletions in the E3 and E4 regions. Obviously, instead of Ad-dl324 virus DNA, strategies of i) and ii) may use DNA of a recombinant virus with a deletion in the E1 region that expresses any transgene to generate a recombinant virus with deletions in the E1, E3, and E4 regions, expressing said transgene.

b) Preparation using cell lines transcomplementing E1 and E4 functions

The essence of this preparation is that a cell line derived from a series expressing the E1 region, for example cell line 293, and also expressing at least the open phases of the OR5 and ORF6 / 7 E4 region of the Ad5 adenovirus under control of a promoter such as an inducible promoter is capable of transcomplementing both the E1 region as well as the E4 region of the Ad5 adenovirus. Such cell lines have been described in Example 4.

Thus, a recombinant virus with deletions in the E1, E3 and E4 regions can be obtained by in vitro ligation or in vivo recombination according to the protocols described below. Whatever protocol was used to generate a virus with a deletion in at least the E4 region, a cytopathic effect (indicative of recombinant virus production) was observed after transfection into the cells used. The cells were then isolated, disrupted by three freeze-thaw cycles in their supernatant and then centrifuged for 10 minutes at 4000 rpm. The supernatant thus obtained was then amplified on fresh cell culture (293 cells for protocols a) and 293 cells expressing the E4 region for b). The viruses were then purified and their DNA analyzed by the method of Hirt (cited above). A virus stock is then prepared on a cesium chloride gradient.

Example 6

This example describes the preparation of defective recombinant adenoviruses of the invention whose genome has deletions of the E1, E3, L5 and E4 genes. These vectors are particularly preferred because the L5 region encodes a chain that is a protein extremely toxic to the cell.

These adenoviruses were prepared from plasmid p2 carrying a modified right part of the genome of the Ad5 adenovirus co-transfected with various helper plasmids. Said adenoviruses may also be prepared using a complementing cell line.

6.1. Construction of plasmid p2

This plasmid contains the entire right region of the Ad5 adenovirus genome starting from the BamHI site (21562), in which a fragment was deleted between the XbaI sites (28592) and AvrII (35463)

-14GB 287157 B6 carrying the E3, L5 and E4 genes. Plasmid p2 was obtained by cloning and ligation of the following fragments in plasmid pIC19R, which was linearized using BamHI and dephosphorylated (Fig. 8):

- an Ad5 genome fragment contained between the BamHI (21562) and XbaI sites (28592), and

- the right end of the Ad5 adenovirus genome (containing the right ITR) from the AvrII site (35463) to the Bell site (BamHI compatible).

6.2 Construction of the helper plasmid (pITRL5-E4) carrying the L5 gene

The helper plasmid pITRL5-E4 carries the E4 and L5 trans genes in the configuration. It corresponds to the plasmid pE4Gal described in Example 2 and additionally contains an L5 coding region under the control of the MLP promoter of the Ad2 adenovirus. Plasmid pITRL5-E4 was constructed as follows (FIGS. 9 and 10).

A 58 bp oligonucleotide containing a HindIII site, an ATG strand, and a coding sequence up to the NdeI site at position 31089 of the Ad5 adenovirus genome is synthesized. The sequence of this oligonucleotide is in the 5'-3 'orientation as follows:

AAGCTTATGAAGCGCGCAAGACCGTCTGAAGATACCTTCAACCCCGTGTATCCATAT G.

HindlII at end 5 'and Ndel at end 3' are underlined once and ATG strings are underlined twice.

The SspI-HindIII fragment containing the MLP promoter sequence followed by the tripartite Ad2 adenovirus leader was isolated from the plasmid pMLPIO (Ballay et al. (1987) UCLA Symposium on Molecular and Cellular Biology, New series, vol. 70, Robinson et al. (Ed.) New). York, 481). This fragment was inserted with the 58 bp oligonucleotide described above between the NdeI and EcoRV sites of plasmid pIC19R to form an intermediate plasmid (see FIG. 9). The SacII- (blunt-ended) -NdeI fragment of plasmid pE4Gal (Example 2) was then introduced into said intermediate plasmid between the SspI and Nddel sites to give plasmid pITRL-E4 (FIG. 10).

6.3. Preparation of defective recombinant adenoviruses containing deletions in the E1, E3, L5 and E4 regions

a) Preparation by co-transfection with helper virus into 293 cells

The essence of this preparation consists in a transcomplementation between a "minivirus" (helper virus) expressing the L5 region or the E4 and L5 regions and a recombinant virus with deletions in at least the E3, E4 and L5 regions.

These viruses were obtained either by in vitro ligation or after in vivo recombination according to the following strategies:

i) Both the Ad-dl324 virus (thimmappaya et al., Cell 31 (1982) 543) and the p2 plasmid digested with BamHI were first ligated in vitro and then transfected together with the helper plasmid pITR5-E4 ( Example 6.2) into 293 cells.

ii) Ad-dl324 virus DNA digested with EcoRI and plasmid p2 digested with BamHI were transfected together with plasmid pITRL5-E4 into 293 cells.

iii) Ad5 adenovirus DNA and plasmid p2 digested with BamHI were ligated and then transfected together with plasmid pITRL5-E4 into 293 cells.

Iv) Ad5 DNA of Ad5 adenovirus digested with EcoRI and plasmid p2 digested with BamHI were transfected together with pITRL5-E4 into 293 cells.

Strategies (i) and (ii) allow the generation of recombinant adenovirus with deletions in the E1, E3, L5 and E4 regions; strategies iii) and iv) allow the generation of recombinant adenovirus with deletions in the E3, L5 and E4 regions. It goes without saying that instead of Ad-dl324 virus DNA, recombinant virus DNAs in the E1 region expressing any transgene can be used in strategies i) and ii) to generate a recombinant virus with deletions in the E1, E3, L5, and E4 regions expressing said transgene .

The above described protocols can also be performed using a helper virus carrying only the L5 region and using a cell line capable of expressing the E1 and E4 regions of the adenovirus described in Example 4.

Alternatively, it is also possible to use a complementing cell line capable of expressing the E1, E4 and L5 regions, thereby completely eliminating the use of said helper virus.

After transfection, the produced viruses are isolated, amplified and purified under the conditions described in Example 5.

Claims (20)

PATENT CLAIMS A defective recombinant adenovirus comprising ITR sequences, an encapsulation sequence, and a heterologous DNA sequence comprising one or more therapeutic genes and / or one or more genes encoding antigenic proteins, wherein said adenovirus is an E1 gene and at least one of the E2, E4 and L1-L5 not working. A defective recombinant adenovirus according to claim 1 of human and / or animal origin. The defective recombinant adenovirus according to claim 2 of human origin, selected from adenoviruses classified in group C, preferably from type 2 or 5 adenoviruses. The defective recombinant adenovirus according to claim 2 of animal origin, selected from adenoviruses of canine, bovine, mouse, sheep, porcine, avian and simian origin. The defective recombinant adenovirus according to any one of the preceding claims, wherein at least the E1 and E4 genes are non-functional. A defective recombinant adenovirus according to any one of the preceding claims, free of late L1-L5 genes. The defective recombinant adenovirus according to claim 1, comprising the ITR sequences, the encapsulating sequence, the heterologous DNA sequence comprising one or more therapeutic genes and / or one or more genes encoding antigenic proteins and a region carrying the E2 gene or a portion thereof. The defective recombinant adenovirus according to claim 1, comprising the ITR sequences, the encapsulating sequence, the heterologous DNA sequence comprising one or more therapeutic B6 genes and / or one or more genes encoding antigenic proteins and a region carrying the E4 gene or a portion thereof. The defective recombinant adenovirus according to claim 1, wherein the genome has a deletion of the E1 and E4 genes. 10. The defective recombinant adenovirus according to claim 1, wherein the genome has a deletion of the E1, L5 and E4 genes. A defective recombinant adenovirus according to any one of the preceding claims, comprising a functional E3 gene under the control of a heterologous promoter. The defective recombinant adenovirus according to claim 1, comprising a heterologous DNA sequence comprising a therapeutic gene selected from genes encoding enzymes, blood derivatives, hormones, limfokines, growth factors, neurotransmitters or precursors thereof, or synthesis enzymes or trophic factors, apolipoproteins, distrofin or minidistrophin, from tumor suppressor genes or from genes encoding factors involved in coagulation. The defective recombinant adenovirus according to claim 1, comprising a heterologous DNA sequence comprising a therapeutic gene which is an antisense gene or sequences whose expression in a target cell allows the expression of genes or transcription of cellular mRNAs to be regulated. The defective recombinant adenovirus according to claim 1, comprising a heterologous DNA sequence comprising a gene encoding an antigenic peptide capable of generating in humans an immune response against microorganisms or viruses. The defective recombinant adenovirus according to claim 14, comprising a heterologous DNA sequence comprising a gene encoding a specific antigen peptide of epstein barr virus, HIV virus, hepatitis B virus, mucosal rabies virus or also a specific antigen peptide of tumors. A defective recombinant adenovirus according to any one of the preceding claims, comprising a heterologous DNA sequence which also comprises sequences allowing expression of the therapeutic gene and / or the gene encoding the antigenic peptide in the infected cell. A defective recombinant adenovirus according to any one of the preceding claims, comprising heterologous DNA which comprises, in front of the therapeutic gene, a signal sequence directing the synthesized therapeutic product into the secretory pathways of the target cell. Pharmaceutical composition, characterized in that it contains at least one defective recombinant adenovirus according to any one of claims 1 to 17 as active ingredient. Pharmaceutical composition according to claim 18, characterized in that it contains a defective recombinant adenovirus according to any one of claims 5 to 10 as the active ingredient. 20. A pharmaceutical composition according to claim 18 or 19 comprising a pharmaceutically acceptable carrier for an injectable formulation.